Yersinia pestis primarily causes a zoonotic disease - plague. Although humans are an accidental, dead-end host, bubonic and pneumonic plague has caused widespread loss of human life during recurrent pandemics. Nearly 2,000 human plague cases occur yearly and small epidemics in South America and Southeast Asia are common. In addition, Y. pestis has great potential for use as a bioterrorism agent. In nature, the bacterium has an obligate flea-mammal (primarily rodent) life cycle. Survival of plague in nature is dependent upon its ability to propagate in fleas and upon its transmission from fleas to mammals. In this proposal we will focus on determinants of the hemin-storage (Hms) phenotype of Y. pestis that are involved in the flea portion of the bacterial life cycle. The Hms + phenotype is required for Y. pestis to cause blockage of the proventricular valve of fleas. Blockage of this valve between the esophagus and stomach of the flea is required for transmission of the disease from fleas to mammals. In vitro the Hms +phenotype is characterized by binding of enormous quantities of exogenous hemin or Congo red (CR) dye to the outer membrane fraction of cells grown at 26- 34[unreadable]C but not at higher temperatures. Thus Hms + cells of Y. pestis incubated at 26[unreadable]C on CR agar form red colonies. We now hypothesize that the Hms phenotype of Y. pestis is the result of an extracellular matrix or biofilm produced by the Hms system. The mechanisms of proventricular blockage, roles of Hms proteins in biofilm formation, and mechanisms of temperature regulation of the phenotype remain to be elucidated. We propose to continue studies on identifying essential hms genes, analyzing the regulation of expression of the Hms + phenotype, as well as characterizing the biochemical properties and physiological role(s) of the Hms system. The specific aims of this proposal are - 1) genetic analysis of essential hms genes; 2) analysis of the regulation of Hms expression; 3) biochemical analysis of the Hms system; and 4) analysis of the physiological role(s) of the Hms system. These studies will enhance our understanding of bacterial features that are required for Y. pestis to block the flea which is essential for the transmission of plague from fleas to mammals. This will facilitate measures to control or eliminate natural reservoirs of plague and control disease transmission. In addition, elucidation of the enzymatic activities of Hms proteins and regulatory mechanisms controlling the synthesis of the extracellular matrix likely produced by the Hms system, may have relevance for biofilm formation by other pathogenic bacteria.